A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening

An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3 '-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3 ' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 x 10-5 U mu L-1 in the concentration scope from 1 x 10-4 U mu L-1 to 1 x 10-1 U mu L-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.

Automated summary

An isothermal, one-pot toolbox based on CRISPR-Cas12a collateral cleavage capability (OPT-Cas) is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. The toolbox allows simple but high-sensitive quantification of TdT activity with low detection limits and achieves extraordinary selectivity. Furthermore, it has been successfully used for the detection of TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells.